This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Thioesters are ubiquitous metabolites and regulators, especially relevant, but not limited, to lipid metabolism. Thioesters are removed through the action of thioesterases, which catalyze hydrolysis reactions to generate parent carboxylic acids and thiols. A majority of these enzymes belong to the hotdog thioesterase family targeted in our studies. The hotdog thioesterase family functions to regulate the levels of acyl-CoA metabolites and acyl-Acyl Carrier Proteins (ACPs) in all forms of life. A wide variety of hotdog thioesterases function within each organism (viz, 21 in humans;29 in Pseudomonas aeruginosa). The hotdog thioesterases were first characterized by biochemists as type II thioesterases, functioning in fatty acid and polyketide synthesis. Despite the fact that they catalyze common chemical reactions, the hotdog fold thioesterases display a wide range of catalytic motifs and substrate recognition elements. Although this diversity provides a targeting advantage for specific inhibitors, it demands the identification, comparative structure analysis, and determination of function and catalytic mechanism for a variety of family members. Our plan is to carry out a detailed study of selected thioesterases in order to elucidate the underlying principles that govern substrate recognition and catalysis, develop a strategy for the design of specific inhibitors, and expand our knowledge of thioester biochemistry. The goal of the study is to determine biochemical functions, catalytic mechanisms and tight binding inhibitors for hotdog thioesterases that are implicated in lipid-related, human genetic disorders that underlie obesity and brain cancer, and that are required for virulence of bacterial pathogens for humans. Our immediate targets include the five E. coli hotdog thioesterases, YbdB, YbcG, YbaW, YicA, Ycil, which are also found in a range of human pathogens. (funded by NIH GM028688).This Hotdog domain is involved in many cellular processes which range from thiester hydrolysis, transcriptional regulation of fatty acid biosynthesis, to the degredation of aromatic compounds such as phenylacetic acid. The "Hotdog fold" consists of seven stranded antiparallel beta-sheets as the "bun", which wraps around a five-turn alpha-helical "sausage". By studying this domain within the thioesterase superfamily we hope to understand the metabolic role it plays as a ubiquitous motif found in all branches of life. In particular we are studying BH1999 thioesterase, 3-HBA CoA thioesterase, and PaaI thioesterase.